We are examining the cooperative interactions between cellular and retrovirus-encoded proteins that together regulate virus replication and alter cell metabolism. These interactions are essential for coordinating virus transcription and mRNA processing and for changing the cellular gene expression program. Research is primarily directed towards identifying the gene products and mechanisms essential for human T-cell leukemia virus type 1 (HTLV-1) infectivity, replication and oncogenic transformation. An infectious molecular clone of HTLV-1 and cell culture systems that support virus replication have been developed. We recently established a single-round infection assay for HTLV-1 which permits an analysis of cell and virus genes required for infection and replication events. We have identified a novel HTLV-1 gene product which is essential for virus replication. We are currently using this assay to examine antiviral agents directed against HTLV-1. In order to define the HTLV-1 gene expression program, we have developed quantitative, real-time RT-PCR methods to monitor viral mRNAs in infected cells. Primary human T-cells have been infected and immortalized with cloned HTLV-1 and with lentivirus vectors that encode HTLV-I regulatory genes in order to examine how cellular signaling networks are reprogrammed by the virus. We have begun to examine HTLV-1 induced alterations in cellular gene expression profiles in primary human T-cells early after infection by DNA microarray analysis. In this regard, we are also investigating HTLV-1 Tax-mediated activation of cellular genes controlled by serum response factor (SRF). We found that transcriptional activation mediated by SRF requires the participation of the cellular cofactors Sap1a and CBP. We are also asking how HTLV-1 modulates src-family tyrosine kinases in T-cells and how these changes affect the T-cell transformation process. We are also comparing the functions and molecular mechanisms of the Rev proteins encoded by various lentiviruses, including human immunodeficiency virus (HIV-1) and its non-primate relatives. This research is focused on a comparative analysis of the components and pathways that regulate retroviral mRNA splicing and transport. These processes are mediated by interactions among the viral Rev proteins, cellular factors, and cis-acting RNA elements. A comparison of HIV-1 and equine infectious anemia virus (EIAV) Rev proteins revealed that the latter mediates alternative splicing and nuclear export of viral pre-mRNA. The distantly related Rev proteins share many features in common but also display distinct differences in the ways in which they interact with RNA. We have characterized the primary RNA element bound by EIAV Rev and showed that it is quite different compared to that of other retroviruses. We have also exploited the EIAV system to identify cellular splicing factors that cooperate with Rev in its post-transcriptional regulation of virus gene expression. We have developed an in vitro RNA splicing system to examine the mechanism of Rev-induced alternative splicing. (Project transferred to BRL) AIDS TITLE: Structure and Function of Lentivirus Tat and Rev Proteins.